The eosinophil-derived neurotoxin (EDN) is a small, glycosylated protein found in the large specific granules of eosinophilic leukocytes. Durack and colleagues (D. T. Durack et at., 1981, Proc. Natl. Acad. Sci., USA 78:5165-5169) reported the isolation of EDN, and determined that eosinophilxe2x80x94related neurotoxicityxe2x80x94a syndrome of ataxia and paralysis associated with Purkinje cell degeneration (the Gordon phenomenon (M. H. Gordon, 1933, Br. Med. J. 1:643)xe2x80x94is mediated in part by the activity of this secretory protein (D. T. Durack et al., 1981, Proc. Natl. Acad. Sci., USA 78:5165-5169; D. T. Durack et al., 1979, Proc. Natl. Acad. Sci., USA 76:1443-1447).
Gleich and colleagues (G. J. Gleich et at., Proc. Natl. Acad. Sci., USA 83:3146-3150) later reported the amino terminal sequence of purified EDN, and noted the similarity between this peptide and the amino-terminal sequence of bovine ribonuclease A (RNase A). EDN""s membership in the RNase A family of ribonuclease genes was later confirmed by molecular cloning (H. F. Rosenberg et al., 1989, Proc. Natl. Acad. Sci., USA 86:4460-4464; R. L. Barker et al., 1989, J. Immunol., 143:952-955). In terms of enzymatic activity, EDN is a catalytically efficient ribonuclease (N. R. Slifman et al., 1986. J. Immunol., 137:2913-2917; U. Gullberg et at, 1986, Biochem. Biophys. Res. Commun., 139:1239-1242; M. Iwami et al., 1981, J. Biochem., 89:1005-1016) and exhibits some degree of preference among experimental substrates (S. Sorrentino et al., 1992, J. Biol. Chem., 267:14859-14865). Both Sorrentino and colleagues (S. Sorrentino et al., 1992, J. Biol. Chem., 267:14859-14865) and Newton and colleagues (D. L. Newton et al., 1994, J. Neurosci., 14:538-544) have shown that EDN""s neurotoxic effects are directly dependent on ribonuclease activity.
While the involvement of eosinophils in the pathophysiology of allergic diseases and asthma has been studied extensively, the potential beneficial roles played by these cells remain poorly understood (A. Sher et al., 1990, Proc. Natl. Acad. Sci. USA, 87:61-65; A. Sher et al., 1990. J. Immunol., 145:3911-3916; F. von Lichtenberg et al., 1985; Am. J. Trop. Med. Hyg., 34:96-106; F. J. Herndon et al., 1992, J. Immunol., 149:3642-3647; C. J. F. Spry 1988. Eosinophils. A comprehensive review and guide to the scientific and medical literature, Oxford University Press, Oxford, UK; G. J. Gleich, 1992, in Inflammation: basic principles and clinical correlates (J. I. Gallin et al., eds.) Raven Press Ltd, New York pp. 663-680). Under physiologic conditions, eosinophils represent only a small fraction of the total leukocytes present in peripheral blood, with the vast majority residing in the perivascular areas of the respiratory and gastrointestinal tracts (C. J. F. Spry, 1988, Oxford University Press, Oxford. UK).
Human eosinophils contain a number of distinct secretory effectors, including eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP) (G. J. Gleich 1992, Raven Press Ltd, New York. pp. 663-680; S. J. Ackerman, 1993 CRC Press, Boca Raton, Fla. pp. 33-70). EDN and ECP are closely-related proteins that have ribonuclease activity (N. R. Slifman et al., 1986, J. Immunol., 137:2913-2917; U. Gullberg et al., 1986, Biophys. Bioch. Res. Commun., 139:1239-1242) and that are members of the ribonuclease A (RNase A) superfamily (G. J. Gleich et al., 1986, Proc. Natl. Acad. Sci. USA. 83:3146-3150; H. F. Rosenberg et al., 1989, J. Exp. Med., 170:163-76; H. F. Rosenberg et al., 1989, Proc. Natl. Acad. Sci., USA 86:4460-4464; H. F. Rosenberg et al., 1995, Nature Genetics 10:219-223).
Although ECP has both anti-parasitic and antibacterial activity in vitro (S. J. Ackerman et al., 1985, Am. J. Trop. Med. Hyg., 34:735-745; R. I. Lehrer et al., 1986, J. Immunol., 142:4428-4434), neither of these functions depends on ribonuclease activity (H. A. Molina et al., 1988, Am. J. Trop. Med. Hyg., 38:327-334; H. F. Rosenberg, 1995, J. Biol. Chem., 270:7876-7881). There is evidence to suggest that ECP destabilizes the lipid membranes of target pathogens (J. D. E. Young et al., 1986, Nature, 321:613-616).
EDN, the major eosinophil ribonuclease, is 100-fold more ribonucleolytically active than ECP (N. R. Slifman et al., 1986, J. Immunol., 137:2913-2917) and although EDN displays specific neurotoxic activity when injected directly into the central nervous systems of experimental animals (D. T. Durack et al., 1979, Proc. Natl. Acad. Sci. USA, 76:1443-1447; D. T. Durack et al., 1981, Proc. Natl. Acad. Sci. USA, 78:5165-5169; D. L. Newton et al., 1994, J. Neurosci., 14:538-544; S. Sorrentino et al., 1992, J. Biol. Chem., 267:14859-14865), it has no defined physiologic function. The ribonucleolytic activities of EDN and ECP, the known membrane-disruptive potential of ECP, and findings relating other ribonucleases to the pathogenesis of viral disease (R. J. Youle et al., 1994, Proc. Natl. Acad. Sci. USA, 91:6012-6016; S. K. Saxena et al., 1996, J. Biol. Chem., 271:20783-20788; A. Mitra et al., 1996, Proc. Natl. Acad. Sci., USA 93:6780-6785; N. M. Cirino et al., 1997, Proc. Natl. Acad. Sci. USA, 94:1937-1942) raise questions about the role of ECP and EDN in the response to infection by enveloped RNA viruses.
Potential target pathogens of EDN and ECP are viruses of the family Paramyxoviridae, including respiratory syncytial virus (RSV) and parainfluenza virus. Although eosinophils are not generally perceived as agents of host defense against viral disease, there are a number of intriguing associations linking eosinophils, eosinophil granule proteins, asthma and allergic bronchospasm, and the pathogenesis of RSV disease (M. C. Seminario and G. J. Gleich, 1994, Curr. Opin. Immunol., 6:860-864; B. Burrows et al., 1977, Am. Rev. Respir. Dis., 115:751-760; B. Zweiman et al., 1996, J. Allerg., 37:48-53; J. T. Twiggs et al., 1981, Clin. Pediatr., 20:187. 190).
RSV has been recognized as the single most important respiratory pathogen in the newborn to two year old age group (C. B. Hall, 1993, Contemporary Pediatrics pp. 92-110). In the United States alone, approximately 67% of all children are infected with RSV within the first year of life, and 50% of those infected develop lower respiratory tract disease. Of this group, 2.5% require hospitalization (approximately 90,000 hospital admissions per year) leading to 4,000 deaths. RSV bronchiolitis in childhood has also been associated with the development of future respiratory disorders, most prominently, Reactive Airways Disease (asthma), a condition currently on the rise in the United States. Recent work has also established the importance of RSV in respiratory compromise in the elderly population (A. R. Falsey et al., 1995, J. Infect. Dis., 172:389-394). Controversy surrounds the use of currently available therapies (Ribavarin, RSV-immune globulin) (C. G. Prober and E. E. L. Wang, 1997, Pediatrics, 99:472-475). There is no vaccine readily available to combat this highly contagious disease.
Several groups have shown that, during RSV infection, eosinophils are recruited to and degranulate into the lung parenchyma (R. Garofalo et al., 1992, J. Pediatr., 120:28-32; E. A. Colocho Zelaya et al., 1994, Ped. All. Immunol., 5:100-106; P. J. Openshaw, 1995, Am. J. Respi. Crit. Care Med., 152.S59-S62; N. Sigurs et al., 1994, Acta Paediatr., 83:1151-1155) and wheezing during RSV infection is associated with increased concentrations of leukotriene C4 (B. Volovitz et al., 1992, J. Pediatr., 112:218-222) and ECP (R. Garofalo et al., 1992, J. Pediatr., 120:28-32) in respiratory secretions. Stark and colleagues (J. M. Stark et al., 1996, J. Immunol., 156:4774-4782) have shown that cultured respiratory epithelial cells infected with RSV support increased adherence of activated eosinophils. Kimpen and colleagues (J. L. L. Kimpen et al., 1995, Pediatric Res. 32:160-164) present evidence suggesting direct activation of eosinophils exposed to RSV in vitro. Saito and colleagues (T. Saito et al., 1997, 175:497-504) have demonstrated that human epithelial cells up-regulate the expression of the eosinophil chemoattractant, RANTES, in response to infection with RSV.
Most dramatically, children previously vaccinated with a formalin-inactivated RSV vaccine who subsequently developed natural RSV infection had increased blood eosinophil counts (J. Chin et al., 1969, Am. J. Epidemiol., 89:449-463) and massive eosinophil infiltrates were observed in post-mortem specimens of vaccinated children who died of RSV pneumonia (H. W. Kim et al., 1969, Am. J. Epidemiol., 89:422-433). These studies demonstrate that recruitment of eosinophils to the respiratory tract can and does occur in response to RSV infection, and, when exaggerated. may lead to a more severe form of RSV disease.
There is a need for agents useful to prevent and treat infection by enveloped RNA viruses, particularly single-stranded RNA viruses such as RSV. The methods and compositions of the present invention address this need.
The present invention provides a method for inactivating a virion of an enveloped RNA virus. The method comprises contacting the virion with an eosinophil-derived ribonuclease. Examples of eosinophil-derived ribonucleases include, but are not limited to, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and congeners thereof. The invention additionally provides a method for treating a subject infected by an enveloped RNA virus. The method comprises administering to the subject an effective amount of an eosinophil-derived ribonuclease, such as EDN or ECP, a congener thereof or a pharmaceutically acceptable salt thereof. The invention additionally provides a method for preventing infection by an enveloped RNA virus in a subject. The method comprises administering to the subject an effective amount of an eosinophil-derived ribonuclease, such as EDN or ECP, a congener thereof or a pharmaceutically acceptable salt thereof.
In one embodiment, the eosinophil-derived ribonuclease is a recombinant protein. In one embodiment, the eosinophil-derived ribonuclease is a human protein. EDN includes proteins comprising the amino acid sequence shown in FIG. 14, SEQ ID NO:7 or 10, or encoded by the nucleotide sequence shown in FIG. 15 (SEQ ID NO:8) or SEQ ID NO:9. ECP includes proteins comprising the coding sequence of human ECP, encoded by nucleotides 55-537 of GenBank Accession No. X15161.
In one embodiment, the enveloped RNA virus is a single-stranded RNA virus, such as a member of the Paramyxoviridae, Orthomyxoviridae, Retroviridae, Togaviridae, Rhabdoviridae, Flaviviridae, Coronaviridae, or Filoviridae families. Members of the Paramyxoviridae family include, but are not limited to, parainfluenza virus (PIV) types 1, 2, 3 and 4, and respiratory syncytical virus (RSV) groups A and B. In another embodiment, the enveloped RNA virus is a double-stranded RNA virus.
The invention further provides a composition comprising an eosinophil-derived ribonuclease, such as eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), a congener thereof, or a pharmaceutically acceptable salt thereof and, optionally, an acceptable carrier. In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the composition is for aerosol administration. In another embodiment, the composition is for parenteral administration.